It is to be noted that throughout this application various publications are referenced by Arabic numerals within brackets. Full citations for these publications are listed at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this invention pertains.
Complement can be activated through either of two distinct enzymatic cascades referred to as the classical and alternative pathways. The classical pathway is usually triggered by antibody bound to a foreign particle, and thus requires prior exposure to that particle for the generation of specific antibody. There are four plasma proteins involved in the classical pathway: C1, C2, C3 and C4. The interaction of C1 with the Fc regions of IgG or IgM in immune complexes activates a C1 protease that can cleave plasma protein C4, resulting in the C4a and C4b fragments. C4b can bind another plasma protein, C2. The resulting species, C4b2, is cleaved by the C1 protease to form the classical pathway C3 convertase, C4b2a. Addition of the C3 cleavage product, C3b, to C3 convertase leads to the formation of the classical pathway C5 convertase, C4b2a3b [1, 2].
In contrast to the classical pathway, the alternative pathway is spontaneously triggered by foreign or other artificial (plastic/glass) or abnormal surfaces (bacteria, yeast, virally infected cells, or damaged tissue), and is therefore capable of an immediate response to an invading organism. There are four plasma proteins directly involved in the alternative pathway: C3, factor B, factor D, and factor P (referred to as ‘properdin’). C3 is converted into C3b and C3a by C3 convertase. Properdin in human blood binds C3b to form P-C3b complex. Factor B binds both free C3b and P-C3b, and both factor B-bound complexes are cleaved by factor D to form a new set of complexes, C3bBb and C3bBbP, which possess C3 convertase activity. The resulting convertase cleaves C3, producing the C3b fragment, which can covalently attach to the target and then interact with factors B and D to form the alternative pathway C3 convertase [3, 4].
The alternative pathway C3 convertase is stabilized by C3b bound properdin. Since the substrate for the alternative pathway C3 convertase is C3, C3 is therefore both a component and a product of the reaction. As the C3 convertase generates increasing amounts of C3b, an amplification loop is established. Furthermore, the classical pathway can also generate C3b that C3b can bind factor B and thereby engage the alternative pathway. This allows more C3b to deposit on a target. Both the classical and alternative pathways converge at C3, which is cleaved to form C3b and C3a. C3a is a potent anaphylatoxin and has been implicated in the pathogenesis of a variety of clinical indications. C3a activates neutrophils, monocytes, platelets, mastcells, and T lymphocytes. C3a has been shown to be important for the induction of paw edema in an adjuvant-induced arthritis model [5, 6].
Addition of C3b to C3 convertase generates C5 convertase, which cleaves C5 to produce C5b and C5a. C5a is the most potent anaphylatoxin that causes alterations in smooth muscle, in vascular tone, and in vascular permeability. It is also a powerful chemotaxin and an activator of neutrophils, monocytes, platelets, endothelial cells, and T lymphocytes. C5a-mediated cellular activation can significantly amplify inflammatory responses by inducing the release of additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachadonic acid metabolites and reactive oxygen species.
The cleavage of Cb produces C5b and C5a. C5b combines with C6, C7, C8, and C9 to form the C5b-9 complex at the surface of the target cell. C5b is also known as the membrane attack complex (MAC). There is now strong evidence that MAC may play an important role in inflammation in addition to its role as a lytic pore-forming complex. C5b-9 is also known to mediate platelet activation. Activated platelets express CD62P (P selectin). P-selectin mediates platelet-monocyte binding, and such binding triggers the release of tissue factor from monocytes. One result of such conjugate formation is the removal of platelets from the circulation, a phenomenon that can contribute to the development of thrombocytopenia.
While complement activation provides a valuable first-line defense against potential pathogens, the activities of complement that promote a protective inflammatory response can also represent a potential threat to the host. For example, C3a and C5a anaphylatoxins recruit and activate neutrophils, monocytes and platelets. These activated cells are indiscriminate in their release of destructive enzymes and may cause organ damage. In addition, complement activation may cause the deposition of lytic complement components resulting in host cell lysis. Currently, there are no approved drugs exist that can inhibit the damages caused by the complement pathway. Based upon the available clinical data, it appears that in most acute injury settings, complement activation is mediated predominantly by the alternative pathway [7]. Therefore, developing suitable methods that inhibit only this pathway without completely obviating the immune defense capabilities would be highly desirable. This would leave the classical pathway intact to handle immune complex processing and to aid in host defense against infection.
Factor B plays a key role in the alternative pathway since it provides the catalytic subunit, Bb, for the C3 convertase, PC3bBb. Since factor B is specific to and is an essential component of the alternative pathway, it presents an attractive target for specifically inhibiting this pathway. Factor B by itself is a zymogen with no known catalytic activity, but after binding PC3b, factor B is cleaved by factor D. Inhibition of factor B results in selective inhibition of factor binding to properdin-bound C3b, thereby leading to the inhibition of formation of C3a, C5a and C5b-9, which are responsible for many deleterious effects mentioned previously. Based on this, it should be possible to develop specific inhibitors or inhibition methods that will (a) prevent factor B binding to properdin-bound C3b, and/or (b) suppress factor B cleavage that prevent Bb generation. Monoclonal antibodies to human factor B have been prepared and tested for their in vitro ability to prevent factor B binding to C3b alone [8]. These antibodies have been tested in endotoxin (LPS) assay and shown to be specific for only gram negative oligosaccharides with relevance to sepsis. Other anti-factor B monoclonal antibodies, although, showed high affinity binding to factor B, stabilized the alternative pathway convertase. The blocking monoclonal antibody inhibited factor B binding to C3b as demonstrated by the inhibition of C3a generation. Surprisingly, none of the prior art publications or patents neither disclose other agents such as peptides, peptidomimetic, oligonucleotides, or any small organic molecules; nor do they suggests the use of factor B inhibitors for the treatment of pathological conditions other than sepsis [8].